Bitespiramycin (primitive name: Shengjimycin) [patent no.: ZL97104440.6] is a derivative of spiramycin formed by isovalerylation of the 4″-position hydroxyl group of the mycarose moiety in spiramycin molecule. It is produced using genetic engineering technology, to generate bacterial genomic combination between the isovaleryl spiramycin gene ist of heat resistant streptomyces and spiramycin. Bitespiramycin has relatively strong activity against Gram-positive bacteria, especially Streptococcus pneumonia, Mycoplasma pneumonia and chlamydia, it exhibits antibacterial activity against erythromycin- and (β-lactam antibiotic-resistant bacteria such as influenza bacillus, Legionella, Clostridium perfringens; it does not exhibit complete cross-resistance with similar drugs. Bitespiramycin has a relatively high lipophicity, its oral absorption is fast with strong permeability into tissues and a wide distribution in the body and a long maintaining time. Therefore it shows a good post-antibiotic effect. Phase III clinical trials for Bitespiramycin produced with genetic engineering technology has been finished, the results showed good therapeutic effect and safety. Application for class I new drug certificate has been put forward for this drug.
Clinic research shows Bitespiramycin is a safe and effective antibiotic. Its therapeutic effect is comparative to antibiotic azithromycin, generally accepted to be the best macrolide currently. Nevertheless, due to the fact that Bitespiramycin is a multi-component drug, for all the efforts to establish tablet production process to be able to obtain products with controllable component proportions and a stable quality, yet the demands on the control of extraction as well as purification processes are relatively rigorous, and the quality inspection process is complex. Meanwhile, to enhance its therapeutic effects, it is necessary to development its injection formulation. It is well known that injections bring about therapeutic effects very quickly, is especially applicable to critically ill patients or those who are not suitable to take the drugs orally. It is certain that injections are more demanding in terms of quality control.
Production of multi-component Bitespiramycin by the corresponding bacteria is stemmed from multi-component characteristics of spiramycin. Being a homogeny of Platenomycin, it is a macrolide antibiotic, its main nucleus being a 16-membered lactone ring. It contains three sugar molecules: forosamine, mycaminose and mycarose, whereas the main component of Bitespiramycin is an isovalerylation derivative of the hydroxyl group in 4″-position of the mycarose moiety in spiramycin molecule. Because the existence of acyltransferase gene in the bacteria producing spiramycin, 3-position of the lactone ring in spiramycin molecule can be acetylated to form spiramycin component II; propionylated to form spiramycin component III; and when the 3-position of the lactone ring is de-acylated, spiramycin component I is formed. Therefore Bitespiramycin is also a mixture of at least three components: isovaleryl spiramycins I, II, III. The structures of three main components of Bitespiramycin are as follows:
wherein:                Isovaleryl spiramycin III R1═COCH2CH3 R2═COCH2CH(CH3)2         Isovaleryl spiramycin II R1═COCH3 R2═COCH2CH(CH3)2         Isovaleryl spiramycin I R1═H R2═COCH2CH(CH3)2         
Researches showed that single components of isovaleryl spiramycin I, II and III all have antibiotic activities, there are no significant differences in their activities [patent application number: 201010119745.1]. According to present quality standard set for Bitespiramycin, the contents of isovaleryl spiramycins (I+II+III) should be no less than 60%. Therefore, a single component (content higher than 60%) can be used to substitute the multi-component mixture. To reduce the production of multi-components, this laboratory constructed bacterium stain WSJ-2 by blocking the 3-O-acyl transferase gene using gene recombination technology in Bitespiramycin generating bacterium WSJ-1. WSJ-2 produced only the isovaleryl spiramycin I [Chunyan Ma et al. Current Microbiology, 2011, 62:16-20]. However, this strain showed very low fermentation unit, it is thus not conducive to large-scale production. It is the intention of this invention to regulate gene acyB2 to modify WSJ-2 strain, to increase the yield of the single component isovaleryl spiramycin I and its content in the mixture. This will lay the foundation for the development of a new drug: single component of the Bitespiramycin homolog, isovaleryl spiramycin I for clinic use.
It is known that the isovaleryltransferase gene ist from heat-resistant streptomyces is linked with regulatory gene acyB2, the presence of the regulatory gene can activate the expression of ist gene. The model streptomyces strain: Variable Lead Green Streptomyces containing complete ist—as well as acyB2-regulatory genes was able to convert 67-79% of the externally added tylosin to 4″-isovaleryl tylosin, whereas the same strain of Streptomyces containing only ist gene, or ist gene with incomplete acyB2 gene, converted only 0-2.4% of the added tylosin [Arisawa A et al: Biosci Biotechnol Biochem 1993, 57(12): 2020-2025]. By transferring recombinant plasmid of autonomous replication form (vectors pIJ702 or pIJ943) containing ist- and acyB2 genes into tylosin producing bacterium (Streptomyces fradiae), under the condition of external addition of a drug (thiopeptin), the transformant mainly produced tylosin in fermentation, besides a minor amount of 4″-isovaleryltylosin (ca. 56 g/mL)[Arisawa A. et al: J Antibiotics 1996,49(4):349-354] could be detected. However, no reports on the increase of 4″-isovaleryltylosin yield or higher content of single component in the mixture by transferring ist- and acyB genes into tylosin producing bacterium have been published up to now.
acyB2 regulatory gene and the transcriptional regulatory protein Srm28c in the spiramycin producing bacterium (Streptomyces ambofaciens) are homologous [Fatma Karray et al Microbiology 2007,153,4111-4122], their degree of identity is 69%, the degree of identity of acyB2 and regulatory protein tylR in tylosin producing bacterium (Streptomyces fradiae) is 41%, tylR is the positive regulatory protein of the biosynthesis in tylosin producing bacterium, it regulates the expression of a polyketide synthase module (tylGI) in tylosin producing bacterium, and plays a regulatory role on the tylosin glycosyl synthesis as well as the polyketone ring oxidation. High expression of tylR gene in the tylosin producing bacterium can increase the yield of tylosin [George S. et al., Mol. Microbiology 2004,54(5):1326-1334]. However, the method of integration of ist gene into the spiramycin producing bacterium, which is homologous as the acyB2, by means of chromosome-recombinant plasmid homologous DNA recombination technology, only resulted in the production of mixture of isovaleryl spiramycin, with a yield of as high as 800 mg/mL [Shang Guangdong et al. Chinese Journal of Biotechnology, 1999,15(2):171]. This demonstrated that only by transferring ist gene into a strain containing regulatory gene similar with acyB2 would not be able to increase significantly the yield and component content of the target product.
This laboratory demonstrated that by concatenating ist gene using genetic engineering technique, to increase the copy number of ist gene in spiramycin producing bacterium to increase ist gene dosage, the capability of the bacteria to isovalerylate was also increased; it was also demonstrated that by substituting the original ist gene promoter sequence with a promoter with strong activity, such as erythromycin resistance gene ermE promoter sequence, the expression of ist gene could be increased. This increases the production of isovaleryl spiramycin by 62% with the gene engineered bacterium [Patent application No. 200910148767.8].
The aim of this invention is to obtain bacterial strains yielding high content of Bitespiramycin single component—isovaleryl spiramycin I, as well as high productivity by means of expression of ist gene linked with regulatory gene acyB2 in a strain, which has already been obtained, capable of producing only the component of isovaleryl spiramycin I, using a highly efficient and stable expression system. To attain this aim, it is necessary to overcome the obstacles of the restriction of the antibiotic producing bacteria of practical value on the gene modification by exogenous gene, thereby improving the conversion rate of the exogenous DNA. Meanwhile, it is necessary to select vector systems for stable express, such that the genetically engineered bacteria can be suitable for large-scale industrial production. This invention proposes to adopt an integrated vector for non-autonomous replication in Streptomyces, such a vector contains the Streptomyces phage integrase enzyme as well as the attachment sites, and contains only the E. coli replicon, to facilitate the preparation in E. coli; meanwhile the vector lacks Streptomyces replicon. When it enters Streptomyces cells, the integration relies on the attachment sites (attP) on the carrier and specific attachment sites of the host bacteria. Therefore, the exogenous gene may be stably expressed in large scale production. The researches on the co-expression of said isovaleryl acylase gene ist and the regulatory gene acyB2 in isovaleryl spiramycin I producing bacterium to increase the yield and content of isovaleryl spiramycin I, as well as the studies on pilot-scale experimental production, have not been found reported to date at home and abroad.